The overall hypothesis to be tested in this proposal is that RNA interference can be developed as a therapeutic modality for HIV-1 disease. RNA interference is a highly evolutionary conserved cellular mechanism whereby small double stranded RNAs induce specific silencing of genes. It is becoming increasingly clear that RNA interference plays a critical role in the regulation of cellular gene expression as well as protection from viruses, most clearly established in plants, and control of retrotransposition. Because RNAi is readily adaptable for knockdown of gene expression any gene for experimental purposes, RNA interference has been proposed as a therapeutic means to inhibit viral replication, including that of HIV-1. During the past funding period of this grant, we successfully developed shRNAs to downregulate CCR5 as a means to inhibit HIV-1 infection. We demonstrated that these shRNAs can downregulate CCR5 in primary T-cells in vitro, and protect cells from HIV-1 infection. As outlined in this proposal, the path towards developing an effective CCR5 shRNA was not straightforward, since it was recognized by ourselves and others in the field that RNAi related toxicity is a major hurdle to in vivo applications, such as HIV-1 disease, where long term expression is essential. Nevertheless, we have successfully overcome these obstacles and developed one shRNA that results in stable downregulation of CCR5. PUBLIC HEALTH RELEVANCE: Relevance RNA interference is a means to block the function of genes. We will investigate whether RNA interference can be used as a potential therapy for HIV-1 disease.